NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation

NOX5 is a ROS-generating NADPH oxidase which contains an N-terminal EF-hand region and can be activated by cytosolic Ca(2+) elevations. However the C-terminal region of NOX5 also contains putative phosphorylation sites. In this study we used HEK cells stably expressing NOX5 to analyze the size and subcellular localization of the NOX5 protein, its mechanisms of activation, and the characteristics of the ROS released. We demonstrate that NOX5 can be activated both by the protein kinase C activating phorbol esther PMA and by the Ca(2+) ionophore ionomycin. The PMA- but not the ionomycin-dependent activation can be inhibited by protein kinase C inhibitors. NOX5 activity is inhibited by submicromolar concentrations of diphenyl iodonium (DPI), but not by apocynin. Western blot analysis showed a lower ( approximately 70 kDa) than expected (82 kDa) molecular mass. Two arguments suggest that NOX5 is at least partially expressed on the plasma membrane: (i) the membrane-impermeant superoxide was readily detected by extracellular probes, and (ii) immunofluorescent labeling of NOX5 detected a fraction of the NOX5 protein at the plasma membrane. In summary, we demonstrate that NOX5 can be found intracellularly and at the cell surface. We also describe that it can be activated through protein kinase C, in addition to its Ca(2+) activation.     

NADPH-Oxidase 2 Promotes Autophagy in Spinal Neurons During the Development of Morphine Tolerance

Neurochemical Research - Repeated morphine administration results in analgesic tolerance. However, the underlying mechanism of morphine analgesic tolerance remains unclear. NADPH-oxidase 2 (NOX2)...     

Uptake of Glycine into Synaptic Vesicles Isolated from Rat Spinal Cord

Glycine was taken up by a synaptic vesicle fraction from spinal cord in a Mg-ATP-dependent manner. The accumulation of glycine was inhibited by carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and nigericin, agents known to destroy the proton gradient across the vesicle membrane. Vesicular uptake of glycine was clearly different from synaptosomal uptake, with respect to both the affinity constant and the effect of Na+, ATP, CCCP, and temperature. Oligomycin and strychnine did not inhibit the vesicular uptake, showing that neither mitochondrial H(+)-ATPase nor binding to strychnine-sensitive glycine receptors was involved. It is suggested that the vesicular uptake of glycine is driven by a proton gradient generated by a Mg2(+)-ATPase. A low concentration of Cl- had little effect on the uptake of glycine, whereas the uptake of glutamate in the same experiment was highly stimulated. High concentrations of gamma-amino-n-butyric acid and beta-alanine inhibited vesicular glycine uptake, but glutamate did not. Accumulation of glycine was found to be fourfold higher in a spinal cord synaptic vesicle fraction than in a vesicle fraction from cerebral cortex.     

Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl

We investigated the mechanism of H(2)O(2) activation of the Ca(2+)-regulated NADPH oxidase NOX5. H(2)O(2) induced a transient, dose-dependent increase in superoxide production in K562 cells expressing NOX5. Confocal studies demonstrated that the initial calcium influx generated by H(2)O(2) is amplified by a feedback mechanism involving NOX5-dependent superoxide production and H(2)O(2). H(2)O(2) NOX5 activation was inhibited by extracellular Ca(2+) chelators, a pharmacological inhibitor of c-Abl, and overexpression of kinase-dead c-Abl. Transfected kinase-active GFP-c-Abl colocalized with vesicular sites of superoxide production in a Ca(2+)-dependent manner. In contrast to H(2)O(2), the Ca(2+) ionophore ionomycin induced NOX5 activity independent of c-Abl. Immunoprecipitation of cell lysates revealed that active GFP-c-Abl formed oligomers with endogenous c-Abl and that phosphorylation of both proteins was increased by H(2)O(2) treatment. Furthermore, H(2)O(2)-induced NOX5 activity correlated with increased localization of c-Abl to the membrane fraction, and NOX5 proteins could be coimmunoprecipitated with GFP-Abl proteins. Our data demonstrate for the first time that NOX5 is activated by c-Abl through a Ca(2+)-mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 NADPH oxidase and c-Abl.     

Turnover of Glial Filaments in Mouse Spinal Cord

Twenty-day-old mice received a single tail vein injection of [guanido-14C]arginine. The cytoskeleton was extracted from the spinal cords at varying lengths of time thereafter. Glial fibrillary acidic protein (GFAP) formed a distinct, broad band that was widely separated from other protein bands in one-dimensional polyacrylamide gels. The purity of the GFAP band was verified by Western blot analysis of one- and two-dimensional electrophoretic patterns. In addition, enzyme-linked immunosorbent assay and quantitative Western blot analysis indicated that 95% of the total spinal cord GFAP was extracted in the cytoskeletal preparation. The specific activity of GFAP was obtained by eluting the protein from the cytoskeletal GFAP band in preparative one-dimensional gels. Specific activity reached a peak 2 h after injection with [14C]arginine. Forty percent of the incorporated radioactivity was still present in cytoskeletal GFAP at 9 weeks, indicating that a significant proportion of glial filaments turns over relatively slowly in vivo.     

Relationship between Spinal Cord Volume and Spinal Cord Injury due to Spinal Shortening

Vertebral column resection is associated with a risk of spinal cord injury. In the present study, using a goat model, we aimed to investigate the relationship between changes in spinal cord volume and spinal cord injury due to spinal shortening, and to quantify the spinal cord volume per 1-mm height in order to clarify a safe limit for shortening. Vertebral column resection was performed at T10 in 10 goats. The spinal cord was shortened until the somatosensory-evoked potential was decreased by 50% from the baseline amplitude or delayed by 10% relative to the baseline peak latency. A wake-up test was performed, and the goats were observed for two days postoperatively. Magnetic resonance imaging was used to measure the spinal cord volume, T10 height, disc height, osteotomy segment height, and spinal segment height pre- and postoperatively. Two of the 10 goats were excluded, and hence, only data from eight goats were analyzed. The somatosensory-evoked potential of these eight goats demonstrated meaningful changes. With regard to neurologic function, five and three goats were classified as Tarlov grades 5 and 4 at two days postoperatively. The mean shortening distance was 23.6 ± 1.51 mm, which correlated with the d-value (post-pre) of the spinal cord volume per 1-mm height of the osteotomy segment (r = 0.95, p < 0.001) and with the height of the T10 body (r = 0.79, p = 0.02). The mean d-value (post-pre) of the spinal cord volume per 1-mm height of the osteotomy segment was 142.87 ± 0.59 mm³ (range, 142.19–143.67 mm³). The limit for shortening was approximately 106% of the vertebral height. The mean volumes of the osteotomy and spinal segments did not significantly change after surgery (t = 0.310, p = 0.765 and t = 1.241, p = 0.255, respectively). Thus, our results indicate that the safe limit for shortening can be calculated using the change in spinal cord volume per 1-mm height.     

Role and Mechanism of Microglial Activation in Iron-Induced Selective and Progressive Dopaminergic Neurodegeneration

Parkinson’s disease (PD) patients have excessive iron depositions in substantia nigra (SN). Neuroinflammation characterized by microglial activation is pivotal for dopaminergic neurodegeneration in PD. However, the role and mechanism of microglial activation in iron-induced dopaminergic neurodegeneration in SN remain unclear yet. This study aimed to investigate the role and mechanism of microglial β-nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) activation in iron-induced selective and progressive dopaminergic neurodegeneration. Multiple primary midbrain cultures from rat, NOX2^+/+ and NOX2^?/? mice were used. Dopaminergic neurons, total neurons, and microglia were visualized by immunostainings. Cell viability was measured by MTT assay. Superoxide (O₂ ^·?) and intracellular reactive oxygen species (iROS) were determined by measuring SOD-inhibitable reduction of tetrazolium salt WST-1 and DCFH-DA assay. mRNA and protein were detected by real-time PCR and Western blot. Iron induces selective and progressive dopaminergic neurotoxicity in rat neuron–microglia–astroglia cultures and microglial activation potentiates the neurotoxicity. Activated microglia produce a magnitude of O₂ ^·? and iROS, and display morphological alteration. NOX2 inhibitor diphenylene iodonium protects against iron-elicited dopaminergic neurotoxicity through decreasing microglial O₂ ^·? generation, and NOX2^?/? mice are resistant to the neurotoxicity by reducing microglial O₂ ^·? production, indicating that iron-elicited dopaminergic neurotoxicity is dependent of NOX2, a O₂ ^·?-generating enzyme. NOX2 activation is indicated by the increased mRNA and protein levels of subunits P47 and gp91. Molecules relevant to NOX2 activation include PKC-σ, P38, ERK1/2, JNK, and NF-КB_P65 as their mRNA and protein levels are enhanced by NOX2 activation. Iron causes selective and progressive dopaminergic neurodegeneration, and microglial NOX2 activation potentiates the neurotoxicity. PKC-σ, P38, ERK1/2, JNK, and NF-КB_P65 are the potential molecules relevant to microglial NOX2 activation.     

Induction of Cholinergic Expression in Developing Spinal Cord Cultures

The induction of choline acetyltransferase (ChAT) by cAMP derivatives was studied in dissociated spinal cord cultures. Dibutyryl cAMP (dbcAMP) and 8-bromo cAMP (1 mM) produced a 2-3-fold stimulation of ChAT activity in developing cultures whereas 8-bromo cGMP had no effect. A phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, also increased (2-fold) ChAT activity in immature cultures. Significant elevations in ChAT were detected after 2 h incubation with dbcAMP. Maximum enzyme induction was observed 24 h after dbcAMP supplementation to the culture medium. Developmental studies revealed that ChAT could be induced on days 2-16 in culture. The largest induction of ChAT activity was observed on day 7 in culture. After day 19, when control enzyme activity attained levels of mature cultures, cAMP-mediated ChAT induction was no longer observed. Cycloheximide and actinomycin D blocked ChAT induction whereas basal enzyme activity remained unaffected. Culture protein content was not changed after 1-day exposure to dbcAMP. 125I-Tetanus toxin fixation after dbcAMP treatment revealed a 20% decrease from control in neuronal surface during days 7-9 in culture. These data indicated that cAMP derivatives produced a rapid increase in cholinergic expression during a specific period of development in spinal cord cultures. There appears to be specificity to this effect, as total neuronal surface does not respond in the same manner as ChAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-Term Changes in Spinal Cord Evoked Potentials After Compression Spinal Cord Injury in the Rat

SUMMARY 1. After traumatic spinal cord injury (SCI), histological and neurological consequences are developing for several days and even weeks. However, little is known about the dynamics of changes in spinal axonal conductivity. The aim of this study was to record and compare repeated spinal cord evoked potentials (SCEP) after SCI in the rat during a 4 weeks’ interval. These recordings were used: (i) for studying the dynamics of functional changes in spinal axons after SCI, and (ii) to define the value of SCEP as an independent outcome parameter in SCI studies.2. We have used two pairs of chronically implanted epidural electrodes for stimulation/recording. The electrodes were placed below and above the site of injury, respectively. Animals with implanted electrodes underwent spinal cord compression injury induced by epidural balloon inflation at Th8–Th9 level. There were five experimental groups of animals, including one control group (sham-operated, no injury), and four injury groups (different degrees of SCI).3. After SCI, SCEP waveform was either significantly reduced or completely lost. Partial recovery of SCEPs was observed in all groups. The onset and extent of recovery clearly correlated with the severity of injury.There was good correlation between quantitated SCEP variables and the volumes of the compressing balloon. However, sensitivity of electropohysiological parameters was inferior compared to neurological and morphometric outcomes.4. Our study shows for the first time, that the dynamics of axonal recovery depends on the degree of injury. After mild injury, recovery of signal is rapid. However, after severe injury, axonal conductivity can re-appear after as long as 2 weeks postinjury.In conclusion, SCEPs can be used as an independent parameter of outcome after SCI, but in general, the sensitivity of electrophysiological data were worse than standard morphological and neurological evaluations.     

Reduced Protein Kinase C Activity in Ischemic Spinal Cord

Protein phosphorylation was evaluated in a rabbit spinal cord ischemia model under conditions where cyclic AMP-dependent protein kinase (PK-A) and calcium/phospholipid-dependent protein kinase (PK-C) were activated. One hour of ischemia did not affect PK-A activity significantly; however, PK-C activity was reduced by more than 60%. In vitro phosphorylation of endogenous proteins by endogenous PK-C revealed that eight particulate and five cytosolic proteins showed stimulated phosphorylation by PK-C activators in control tissue, although this stimulation was virtually absent in ischemic samples. When control and ischemic particulate fractions were combined, the endogenous protein phosphorylation pattern under PK-C-activating conditions was similar to the ischemic sample, which suggests that inhibitory molecules may be present in the ischemic particulate fraction. In vitro phosphorylation of endogenous proteins under PK-A-activating conditions in ischemic tissue was similar to that in control tissue. The results suggest that the PK-C phosphorylation system is selectively impaired in ischemic spinal cord. In addition to reduced PK-C-dependent phosphorylation, an Mr 64,000 protein was phosphorylated in ischemic cytosolic samples, but not in control samples. The phosphorylation of the Mr 64,000 protein was neither PK-C-dependent nor PK-A-dependent. These altered phosphorylation reactions may play critical roles in neuronal death during the course of ischemia.     

Serum Lipid Profile in Subjects with Traumatic Spinal Cord Injury

ResultsCases without preserved motor function (AIS A or B) had lower total and HDL cholesterol than the others (-11.4 [-21.5, -1.4] mg/dL total cholesterol and -5.1 [-8.8, -1.4] mg/dL HDL-c), and cases with all-limb involvement had lower total, HDL, and LDL cholesterol than those with only lower-limb involvement (-14.0 [-24.6, -3.4] mg/dL total cholesterol, -4.1 [-8.0, -0.2] mg/dL HDL-c, and -10.0 [-19.7, -0.3] mg/dL LDL-c) (all p<0.05). No association was found between lipid concentrations and time since injury. Concentrations of lipid subfractions and triglycerides in SCI subjects were lower than in sex- and age-stratified values from the reference sample.ConclusionA high degree of neurological involvement in SCI (anatomically higher lesions and AIS A or B) is associated with lower total cholesterol and HDL-c.     

Brain and Spinal Cord Lesions in Encephalitozoonosis in Mink

Central nervous system lesions were studied by light microscopy in 43 farmed mink, aged 5 months to 2 1/2 years, with spontaneous encephalitozoonosis and showing cataractous eye changes. Lesions were found in the brain and spinal cord of all animals examined but were generally mild and chronic. The lesions were consistent with those previously described in spontaneous encephalitozoonosis in other carnivores. Parasites in parasitophorous vacuoles and free or phagocytosed in necrotic and granulomatous lesions were demonstrated in animals aged 5 months to 1 year. The occurrence of arterial lesions of the polyarteritis nodosa type found in the youngest animals probably indicates fetal infection. In animals aged 1 1/2 and 2 1/2 years active lesions were usually lacking and the changes were characterized by arterial sclerosis, sometimes with aneurysmal formations, small perivascular lympho-plasmacytic cuffings and focal gliosis.     

NADH Oxidase Functions as an Adhesin in Streptococcus pneumoniae and Elicits a Protective Immune Response in Mice

The initial event in disease caused by S. pneumoniae is adhesion of the bacterium to respiratory epithelial cells, mediated by surface expressed molecules including cell-wall proteins. NADH oxidase (NOX), which reduces free oxygen to water in the cytoplasm, was identified in a non-lectin enriched pneumococcal cell-wall fraction. Recombinant NOX (rNOX) was screened with sera obtained longitudinally from children and demonstrated age-dependent immunogenicity. NOX ablation in S. pneumoniae significantly reduced bacterial adhesion to A549 epithelial cells in vitro and their virulence in the intranasal or intraperitoneal challenge models in mice, compared to the parental strain. Supplementation of Δnox WU2 with the nox gene restored its virulence. Saturation of A549 target cells with rNOX or neutralization of cell-wall residing NOX using anti-rNOX antiserum decreased adhesion to A549 cells. rNOX-binding phages inhibited bacterial adhesion. Moreover, peptides derived from the human proteins contactin 4, chondroitin 4 sulfotraferase and laminin5, homologous to the insert peptides in the neutralizing phages, inhibited bacterial adhesion to the A549 cells. Furthermore, rNOX immunization of mice elicited a protective immune response to intranasal or intraperitoneal S. pneumoniae challenge, whereas pneumococcal virulence was neutralized by anti-rNOX antiserum prior to intraperitoneal challenge. Our results suggest that in addition to its enzymatic activity, NOX contributes to S. pneumoniae virulence as a putative adhesin and thus peptides derived from its target molecules may be considered for the treatment of pneumococcal infections. Finally, rNOX elicited a protective immune response in both aerobic and anaerobic environments, which renders NOX a candidate for future pneumococcal vaccine.     

Lipid and Protein Alterations of Spinal Cord and Cord Myelin of Multiple Sclerosis

Abstract: A comprehensive study was carried out to clarify the chemical compositions of spinal cord, cord myelin, and myelin subfractions of multiple sclerosis (MS). The protein compositions of normal-appearing cerebral white matter and cerebral plaque and periplaque tissues were also analyzed for comparison. MS whole cord samples were found to contain higher amounts of water compared with normal samples. The total lipid contents were below normal. Among the individual lipids, cholesterol content remained unchanged, whereas cholesteryl esters appeared increased in MS cords. The acidic phospholipid concentrations were found to be lower than normal. Glycolipids, such as cerebrosides G_M4, G_M1, and G_D1b, which are abundant in myelin, were all decreased. However, the concentrations of G_M3 and G_D3, which are more characteristic of reactive astrocytes, were highly elevated. The total protein content of MS cord samples was decreased, and the decrease was attributable to the loss of myelin proteins as evidenced by the low recovery of myelin. The concentrations of myelin-specific proteins, such as proteolipid protein and myelin basic protein, were significantly reduced. Other changes in the protein compositions included the accretion of two low molecular weight proteins of approximately 11,000 and 12,000, and the appearance of a periodic acid-Schiff-positive protein with the same electrophoretic mobility as the P₀ protein. Analysis of the isolated myelin indicated that it had a grossly normal protein composition. However, the two low molecular weight proteins and the P₀ protein appeared to be enriched in an upper-phase cord subtraction. We attribute the appearance of the two low molecular weight proteins to the breakdown of proteolipid protein and/or myelin basic protein as a result of demyelination, and the appearance of P₀ to the involvement of PNS myelin. The latter finding provides the first biochemical evidence that in MS cord, remyelination can be achieved in part by invading Schwann cells and/or by the small number of Schwann cells that may be present in the cord.     

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.Open in a separate windowClick here to view.^{(19M, flv)}     

Involvement of NADPH oxidase in up-regulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts induced by oxidized LDL and in apolipoprotein E-deficient mice

The present study demonstrated that oxidized LDL (oLDL) increased the generation of superoxide and hydrogen peroxide (H(2)O(2)), the abundances of NADPH oxidase (NOX)4, NOX2, p22-phox and lectin-like oLDL receptor-1 (LOX-1) in wild-type or heat shock factor-1 (HSF1)-deficient mouse embryo fibroblasts (MEF). LOX-1 antibody inhibited LDL or oLDL-induced expression of NOX components in MEF. Abundance of HSF1 or plasminogen activator inhibitor-1 (PAI-1) was increased by oLDL in wild-type, but not in HSF1-deficient MEF. Diphenyleneiodonium or siRNA for NOX or p22-phox inhibited oLDL-induced increases of HSF1, PAI-1 and H(2)O(2) in MEF. Increased NOX4, NOX2, LOX1, HSF1 and PAI-1 were detected in aortae and hearts of apolipoprotein E-knockout (apoE-KO) mice compared to controls, which were associated with increased serum cholesterol or plasma PAI-1. The results suggest that NOX is required for oLDL-induced HSF1 or PAI-1 expression in MEF, which was supported by the up-regulation of NOX, LOX-1, HSF1 and PAI-1 in apoE-KO mice.     

器官型培养的脊髓与体内脊髓的对比

旨在观察体外器官型培养的脊髓薄片是否与同龄大鼠体内生长的脊髓具有相似的形态和恒定的前角a运动神经元数目,建立能模拟体内生长环境的稳定的脊髓器官培养模型。利用出生后8天乳鼠的腰段脊髓组织切片建立脊髓器官型培养模型,用神经元的特异性免疫组化染色SMI-32对脊髓前角a运动神经元加以鉴定并与同龄大鼠体内生长的脊髓做比较。结果发现脊髓体外生长良好,形态完整,a运动神经元数目恒定,与同龄大鼠比较无显著差异,并可长期存活达2个月。脊髓的器官培养技术为研究脊髓生理、病理改变及神经保护提供了有效的方法。     

Microvessels from the Spinal Cord: Isolation Procedure and Characterization of the Fraction

Abstract: A simple method for the preparation of microvessels from the spinal cord is described. The fraction being enriched in the microvessels is characterized by morphological techniques and compared biochemically with the original suspension. The method makes possible detailed studies on the metabolism of microvessels and can be complementary to procedures that have been developed for separating the capillaries from other brain tissues.     

NOX1 Supports the Metabolic Remodeling of HepG2 Cells

NADPH oxidases are important sources of reactive oxygen species (ROS) which act as signaling molecules in the regulation of protein expression, cell proliferation, differentiation, migration and cell death. The NOX1 subunit is over-expressed in several cancers and NOX1 derived ROS have been repeatedly linked with tumorigenesis and tumor progression although underlying pathways are ill defined. We engineered NOX1-depleted HepG2 hepatoblastoma cells and employed differential display 2DE experiments in order to investigate changes in NOX1-dependent protein expression profiles. A total of 17 protein functions were identified to be dysregulated in NOX1-depleted cells. The proteomic results support a connection between NOX1 and the Warburg effect and a role for NOX in the regulation of glucose and glutamine metabolism as well as of lipid, protein and nucleotide synthesis in hepatic tumor cells. Metabolic remodeling is a common feature of tumor cells and understanding the underlying mechanisms is essential for the development of new cancer treatments. Our results reveal a manifold involvement of NOX1 in the metabolic remodeling of hepatoblastoma cells towards a sustained production of building blocks required to maintain a high proliferative rate, thus rendering NOX1 a potential target for cancer therapy.